Solid phase synthesis of peptides containing the non-hydrolysable analog of (O)phosphotyrosine, p(CH2PO3H2)Phe. Application to the synthesis of 344-357 sequences of the beta 2 adrenergic receptor.

International audienceStudies about phosphorylation-dephosphorylation mechanisms require the development of probes capable of being used in in vitro and in vivo conditions. We show in this work that the chemically and enzymatically stable p(CH2PO3H2)Phe analog of (O)phosphotyrosine can be easily introduced in peptides by the solid-phase method. It has been incorporated in the 344-357 sequence of the beta 2 adrenergic receptor in place of the Tyr residue in position 350 and/or 354 in order to investigate the role of tyrosine phosphorylation in the receptor agonist-induced down-regulation. Since p(CH2PO3H2)Phe is an ionized hydrophilic residue, peptides containing this amino acid do not easily permeate the cellular membranes. Therefore the modified amino acid was introduced in the synthetic pathway in its N-Boc-p(CH2PO3Et2)Phe form, which could be partially or completely deprotected. Coupling steps, including that of the new amino acid, were performed with good yields (approximately 60% total yield) and further deprotections provided both the p(CH2PO3H2)Phe and p(CH2PO3HEt)Phe containing peptides with yields of around 20% each. The structure of the peptides was assessed by NMR, mass spectroscopy and amino acid analysis and the new amino acid was characterized under its phenylthiocarbamyl form (PTC).Studies about phosphorylation-dephosphorylation mechanisms require the development of probes capable of being used in in vitro and in vivo conditions. We show in this work that the chemically and enzymatically stable p(CH2PO3H2)Phe analog of (O)phosphotyrosine can be easily introduced in peptides by the solid-phase method. It has been incorporated in the 344-357 sequence of the beta 2 adrenergic receptor in place of the Tyr residue in position 350 and/or 354 in order to investigate the role of tyrosine phosphorylation in the receptor agonist-induced down-regulation. Since p(CH2PO3H2)Phe is an ionized hydrophilic residue, peptides containing this amino acid do not easily permeate the cellular membranes. Therefore the modified amino acid was introduced in the synthetic pathway in its N-Boc-p(CH2PO3Et2)Phe form, which could be partially or completely deprotected. Coupling steps, including that of the new amino acid, were performed with good yields (approximately 60% total yield) and further deprotections provided both the p(CH2PO3H2)Phe and p(CH2PO3HEt)Phe containing peptides with yields of around 20% each. The structure of the peptides was assessed by NMR, mass spectroscopy and amino acid analysis and the new amino acid was characterized under its phenylthiocarbamyl form (PTC)

Preferential conformation of the endogenous opiate-like pentapeptide met-enkephalin in DMSO-d<SUB>6</SUB> solution determined by high field <SUP>1</SUP>H NMR

A preferential conformation of the endogenous opiate-like pentapeptide Met-Enkephalin in DMSO-d6 solution was proposed from high field 1H NMR experiments at variable temperature and complete analysis of the coupling constants in relation with conformational energy steric maps. This conformation is characterized by a highly folded secondary structure with a &#946;I turn involving a head-to-tail interaction and a quasi-axial position of the methionine side chain. The N-terminal Tyr-Gly moiety which exhibits a relative degree of freedom shows all the steric requirements found in opiates for a stereospecific interaction with the receptor. All these results are discussed in relation with the physicochemical and biological properties of opiate-like peptides

Acetaldehyde-enkephalins: structure proof and some conformational deductions from one- and two-dimensional proton nuclear magnetic resonance spectra

The structure of the adduct formed by reaction of acetaldehyde and Mets-enkephalin has been determined by analysis of 400-MHz proton spectra: two-dimensional J spectroscopy was used to resolve and measure virtually all the overlapping resonances, and decoupling difference spectroscopy was used to assign the resonances. Suitable manipulation of the two-dimensional data allowed analysis of a-CH resonances which were completely buried under a water signal and of amide NH resonances which overlapped in both dimensions. The adduct was shown to be a mixture of two diastereoisomers, each containing a 2-methylimidazolidin-4-one ring formed by condensation of an acetaldehyde molecule with the N-terminal amino group and Gly2 amide nitrogen. Analysis of the NMR data suggests that the folded conformation characteristic of native enkephalins in dimeth~1-dsu~l foxide is not important in these derivatives